T Cells in Multisystem Inflammatory Syndrome in Children (MIS-C) Have a Predominant CD4+ T Helper Response to SARS-CoV-2 Peptides and Numerous Virus-Specific CD4− CD8− Double-Negative T Cells

We studied SARS-CoV-2-specific T cell responses in 22 subacute MIS-C children enrolled in 2021 and 2022 using peptide pools derived from SARS-CoV-2 spike or nonspike proteins. CD4+ and CD8+ SARS-CoV-2-specific T cells were detected in 5 subjects, CD4+ T helper (Th) responses alone were detected in 12 subjects, and CD8+ cytotoxic T cell (CTL) responses alone were documented in 1 subject. Notably, a sizeable subpopulation of CD4− CD8− double-negative (DN) T cells out of total CD3+ T cells was observed in MIS-C (median: 14.5%; IQR 8.65–25.3) and recognized SARS-CoV-2 peptides. T cells bearing the Vβ21.3 T cell receptor (TcRs), previously reported as pathogenic in the context of MIS-C, were detected in high frequencies, namely, in 2.8% and 3.9% of the CD4+ and CD8+ T cells, respectively. However, Vβ21.3 CD8+ T cells that responded to SARS-CoV-2 peptides were detected in only a single subject, suggesting recognition of nonviral antigens in the majority of subjects. Subjects studied 6–14 months after MIS-C showed T cell epitope spreading, meaning the activation of T cells that recognize more SARS-CoV-2 peptides following the initial expansion of T cells that see immunodominant epitopes. For example, subjects that did not recognize nonspike proteins in the subacute phase of MIS-C showed good Th response to nonspike peptides, and/or CD8+ T cell responses not appreciable before arose over time and could be detected in the 6–14 months’ follow-up. The magnitude of the Th and CTL responses also increased over time. In summary, patients with MIS-C associated with acute lymphopenia, a classical feature of MIS-C, showed a physiological response to the virus with a prominent role for virus-specific DN T cells.


Introduction
A unique feature of the SARS-CoV-2 pandemic has been the emergence of a new syndrome in children exposed to SARS-CoV-2 who present 4 to 6 weeks later with fever and severe systemic inflammation requiring admission to an intensive care unit [1]. Multisystem inflammatory syndrome in children (MIS-C) [2,3] has been clinically well defined, but the pathogenesis is still debated [4][5][6].

Detection of SARS-CoV-2 CD4+ and CD8+ T Cell Responses in MIS-C Subjects
We enrolled 22 MIS-C subjects (Table 1) to study the T cell response to spike and nonspike SARS-CoV-2 peptide pools.
Peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with different peptide megapools tailored to capture T helper (Th) CD4+ T cell responses and cytotoxic CD8+ T cell (CTL) responses. CD4+ T cell responses were evaluated using the AIM assay by measuring by flow cytometry the expression of two costimulatory molecules, the tumor necrosis factor receptor superfamily member 4, also known as CD134 or OX40 receptor, and the tumor necrosis factor ligand superfamily member 9, also known as 4-1BB, 24 h after incubation of PBMC cultures with peptide megapools. CD8+ T cell responses were evaluated by measuring the expression of 4-1BB and CD69 24 h after incubation of PBMC cultures with peptide megapools.
Together, only 6/22 (27%) subjects had SARS-CoV-2-specific CTL in circulation. The CD4+ Th response was significantly higher than the CTL response in the MIS-C cohort ( Figure 1, panel B). C-C chemokine receptor 6 (CCR6), which determines T cell homing to the endothelial and mucosal sites, was expressed with a different magnitude on AIM+ CD4+ and CD8+ T cells (Figure 1, panel C).

Expression of Memory Markers and Chemokine Receptors in SARS-CoV-2-Specific T Cells in MIS-C
Next, we characterized SARS-CoV-2-specific terminally differentiated effector T cells (TEMRA), effector memory T cells (TEM), and central memory T cells (TCM) by measuring the CD45RA and CCR7 expression on AIM+ T cells. SARS-CoV-2-specific CD4+ T cells were of the TEM and TCM subsets, and a low percentage of TEMRA cells were detected ( Figure 2, upper panel). The T cell memory repertoire within SARS-CoV-2-specific CD8+ T cells was different. TEMRA cells were measurable in 6 subjects, and TEM and TCM cells were greater than 20% in 3 subjects and not detectable in 5 subjects despite the coordinated CD4+ T cell response ( Figure 2, lower panels).

Expression of Memory Markers and Chemokine Receptors in SARS-CoV-2-Specific T Cells in MIS-C
Next, we characterized SARS-CoV-2-specific terminally differentiated effector T cells (TEMRA), effector memory T cells (TEM), and central memory T cells (TCM) by measuring the CD45RA and CCR7 expression on AIM+ T cells. SARS-CoV-2-specific CD4+ T cells were of the TEM and TCM subsets, and a low percentage of TEMRA cells were detected ( Figure 2, upper panel). The T cell memory repertoire within SARS-CoV-2-specific CD8+ T cells was different. TEMRA cells were measurable in 6 subjects, and TEM and TCM cells were greater than 20% in 3 subjects and not detectable in 5 subjects despite the coordinated CD4+ T cell response ( Figure 2, lower panels).  (1, 2, 5, 18, and 19); blue circles: subjects responding to both CD4 spike and nonspike epitopes (3,8,9,17, and 21); white circle: subject responding to either CD4 spike or nonspike epitopes (4, 10-12, 15, 20, and 22); gray circle: subject responding to only CD8 epitopes (7). Each symbol represents data derived from each individual subject. SARS-CoV-2specific CD4+ T cells showed higher percentages of TEM and TCM phenotypes and lower percentages of TEMRA than SARS-CoV-2-specific CD8+ T cells.

CD4− CD8− Double-Negative (DN) T Cells Are Numerous in Circulation and Respond to SARS-CoV-2 Peptides
When we gated on CD3+ T cells in peptide-stimulated PBMC cultures to define AIM+ CD4+ and CD8+ T cells, we noted that a high percentage of the T cells (>40% in some subjects) were CD3+ but DN T cells that did not express CD4 or CD8 coreceptors (Supplementary Figure S1). To characterize this population, we first defined the ability of DN T cells to recognize the SARS-CoV-2 megapools determining the AIM+ T cells under stimulatory conditions. The results revealed that DN T cells are fully functional and respond to peptides with a greater stimulation index than canonical T cells ( Figure 3, panel A). Subjects who showed a CD4+ T cell response to peptides, namely, subjects 2, 4, 5, 8, 9, 12,15, 19, and 22, had in circulation DN T cells that recognized CD4 megapools. Two subjects, 14 and 16, whose single positive CD4+ and CD8+ T cells did not respond to peptides, showed a great DN response to CD4 and CD8 megapools ( Figure 3, panel A).
mentary Figure S1). To characterize this population, we first defined the ability of DN T cells to recognize the SARS-CoV-2 megapools determining the AIM+ T cells under stimulatory conditions. The results revealed that DN T cells are fully functional and respond to peptides with a greater stimulation index than canonical T cells ( Figure 3, panel A). Subjects who showed a CD4+ T cell response to peptides, namely, subjects 2, 4, 5, 8, 9, 12,15, 19, and 22, had in circulation DN T cells that recognized CD4 megapools. Two subjects, 14 and 16, whose single positive CD4+ and CD8+ T cells did not respond to peptides, showed a great DN response to CD4 and CD8 megapools ( Figure 3, panel A).
CCR6 was also expressed on DN T cells that recognized CD4 and CD8 peptide pools, suggesting their possible homing to endothelial sites or the gut ( Figure 3, panel B). The expression of programmed cell death protein-1 (PD1) correlated with the expression of the IL-2 receptor CD25, therefore suggesting that these DN T cells are activated rather than exhausted ( Figure 3, panel C). It also appeared that DN T cells did not become CD4+ CD8+ double positive (DP) as the DP T cells in circulation were very few and did not respond to SARS-CoV-2 peptides (Supplementary Figure S2, panel A and B).  CCR6 was also expressed on DN T cells that recognized CD4 and CD8 peptide pools, suggesting their possible homing to endothelial sites or the gut (Figure 3, panel B). The expression of programmed cell death protein-1 (PD1) correlated with the expression of the IL-2 receptor CD25, therefore suggesting that these DN T cells are activated rather than exhausted ( Figure 3, panel C). It also appeared that DN T cells did not become CD4+ CD8+ double positive (DP) as the DP T cells in circulation were very few and did not respond to SARS-CoV-2 peptides (Supplementary Figure S2, panel A and B).

Enumeration and SARS-CoV-2 Response by Vβ21.3 T Cells
In an effort to understand MIS-C pathogenesis, a specific T cell receptor β chain, Vβ21.3, has been linked to the inflammatory process [11,12]. We studied the SARS-CoV-2 response by Vβ21.3 CD4+ and CD8+ T cells in 6 MIS-C subjects (1,18,19,20,21,22) and confirmed previous observations that this TcR rearrangement is relevant in MIS-C with this receptor expressed in up to 2.7% of the CD4+ T cells and 3.9% of the CD8+ T cells (Figure 4, panel A and Supplementary Figure S3

Enumeration and SARS-CoV-2 Response by Vb21.3 T Cells
In an effort to understand MIS-C pathogenesis, a specific T cell receptor b chain, Vb21.3, has been linked to the inflammatory process [11,12]. We studied the SARS-CoV-2 response by Vb21.3 CD4+ and CD8+ T cells in 6 MIS-C subjects (1,18,19,20,21,22) and confirmed previous observations that this TcR rearrangement is relevant in MIS-C with this receptor expressed in up to 2.7% of the CD4+ T cells and 3.9% of the CD8+ T cells (Figure 4, panel A and Supplementary Figure S3

Immune Phenotype of the Antigen Presenting Cells in MIS-C
Next, we enumerated and determined the maturation/activation state of monocytes, macrophages, and myeloid dendritic cells, including cDC1, CD14+ cDC2, CD14−, cDC2, pediatric CD4+ ILT-4+ tolerogenic DC (tmDC), and plasmacytoid DC (pDC), in the 22 subjects studied for SARS-CoV-2-specific T cell responses. CD4+ ILT-4+ tmDC is important in controlling pediatric immune homeostasis [14,15]. In MIS-C, the status of the innate compartment and the extent of the activation of tolerogenic CD14+ cDC2 and tmDC could have played a significant role in the disease pathogenesis. The results, shown in Figure 5, revealed that CD14+ cDC2 and tmDC, both suppressive myeloid lineages, were numerous in circulation and up to 100% CD123+ and 80% CD86+, suggesting both maturity and activation. All the other lineages were within physiological ranges, including pDC, important in the innate response to viruses. Next, we enumerated and determined the maturation/activation state of monocytes, macrophages, and myeloid dendritic cells, including cDC1, CD14+ cDC2, CD14−, cDC2, pediatric CD4+ ILT-4+ tolerogenic DC (tmDC), and plasmacytoid DC (pDC), in the 22 subjects studied for SARS-CoV-2-specific T cell responses. CD4+ ILT-4+ tmDC is important in controlling pediatric immune homeostasis [14,15]. In MIS-C, the status of the innate compartment and the extent of the activation of tolerogenic CD14+ cDC2 and tmDC could have played a significant role in the disease pathogenesis. The results, shown in Figure 5, revealed that CD14+ cDC2 and tmDC, both suppressive myeloid lineages, were numerous in circulation and up to 100% CD123+ and 80% CD86+, suggesting both maturity and activation. All the other lineages were within physiological ranges, including pDC, important in the innate response to viruses.

Expansion of CD4+ Th Cells and T Cell Memory in MIS-C Studied 6-14 Months after MIS-C Onset
Of the 22 MIS-C subjects, 6 (27%) (2, 3, 5, 12, 14, and 16) were studied for their SARS-CoV-2 T cell responses in their subacute phase and again 6-14 months later. The magnitude of the CD4+ Th cell responses to the spike proteins increased in 4 subjects (5, 12, 14, and 16) (Figure 6, panel A). T cells from subjects 5, 12, 14, and 16 that did not respond to nonspike CD4 megapools in the subacute phase of MIS-C showed a response in the follow-up visit, suggesting epitope spreading over time (Figure 6, panel A). Epitope spreading included CD8+ T cell responses in subjects 2 and 3 at the follow-up visit (Figure 6, panel A). The distribution of CCR6 on CD4+ and CD8+ T cells was similar to what we observed in the subacute phase in all the 6 subjects studied (Figure 6, panel A). Within the development of T cell memory, TEMRA cells were absent in the CD4 compartment but numerous within CD8+ T cells (Figure 6, panel B). CD4+ TEM increased from the subacute phase, showing increased T cell memory to spike and nonspike proteins ( Figure 6, panel B). DN T cells were still numerous in circulation, and as in the subacute phase, subjects 3, 14, and 16 recognized SARS-CoV-2 CD4 peptides ( Figure 6, panel C). Interestingly, Vβ21.3 T cells were still present several months (2.0-3.9% of CD4+ T cells; 0.9-2.6% of CD8+ T cells) after the acute illness and, as in the subacute phase, did not respond to SARS-CoV-2 peptides ( Figure 6, panel D).

Expansion of CD4+ Th Cells and T Cell Memory in MIS-C Studied 6-14 Months after MIS-C Onset
Of the 22 MIS-C subjects, 6 (27%) (2, 3, 5, 12, 14, and 16) were studied for their SARS-CoV-2 T cell responses in their subacute phase and again 6-14 months later. The magnitude of the CD4+ Th cell responses to the spike proteins increased in 4 subjects (5, 12, 14, and 16) (Figure 6, panel A). T cells from subjects 5, 12, 14, and 16 that did not respond to nonspike CD4 megapools in the subacute phase of MIS-C showed a response in the follow-up visit, suggesting epitope spreading over time (Figure 6, panel A). Epitope spreading included CD8+ T cell responses in subjects 2 and 3 at the follow-up visit (Figure 6, panel A). The distribution of CCR6 on CD4+ and CD8+ T cells was similar to what we observed in the subacute phase in all the 6 subjects studied (Figure 6, panel A). Within the development of T cell memory, TEMRA cells were absent in the CD4 compartment but numerous within CD8+ T cells (Figure 6, panel B). CD4+ TEM increased from the subacute phase, showing increased T cell memory to spike and nonspike proteins ( Figure 6, panel  B). DN T cells were still numerous in circulation, and as in the subacute phase, subjects 3, 14, and 16 recognized SARS-CoV-2 CD4 peptides ( Figure 6, panel C). Interestingly, Vb21.3 T cells were still present several months (2.0%-3.9% of CD4+ T cells; 0.9%-2.6% of CD8+ T cells) after the acute illness and, as in the subacute phase, did not respond to SARS-CoV-2 peptides.  Figure 6. T cell responses to peptide megapools derived from SARS-CoV-2 in MIS-C subjects healthy 6-14 months after the disease onset. Six MIS-C subjects (2, 3, 5, 12, 14, and 16) studied at their subacute phase were also studied 6-14 months later. Canonical CD4+ and CD8+, CD4− CD8− DN, and TCR Vβ21.3+ CD4+ and CD8+ T cell responses to SARS-CoV-2 peptide megapools were studied. The SI of AIM+ T cells from each individual subject was calculated to study the T cell responses. (A) CD4+ and CD8+ T cell responses to peptide megapools and their CCR6 expressions. Three subjects (2, 3, and 5) of the 6 subjects showed concurrent CD4+ and CD8+ T cell responses, and the other 3 follow-up MIS-C subjects (12, 14, and 16) showed only CD4+ T cell responses to SARS-CoV-2 (both CD4 spike (S) and nonspike (R) megapools). (B) Memory phenotypes of AIM+ CD4+ (left panels) and CD8+ (right panels) T cells. Red circles: subjects responding to both CD4 and CD8 epitopes (subjects 2, 3, and 5); blue circles: subjects responding to both CD4 spike and nonspike epitopes (subjects 12, 14, and 16). AIM+ CD4+ T cells showed a similar level of TEMRA, TEM, and TCM at follow-up visit compared with subacute MIS-C subjects (p > 0.05). AIM+ CD8+ T cells showed a slight increase in TEM (p > 0.05) and a similar level of TEMRA and TCM. (C) Percentage of DN T cells (left panel) and their responses to SARS-CoV-2 peptide megapools (right panels). Percentages of DN T cells were decreased in 5 subjects (2, 3, 5, 12, and 14) compared with subacute MIS-C subjects. One subject (3) showed DN T cell responses to SARS-CoV-2 CD4 and CD8 peptide megapools, and 2 subjects (14 and 16) showed DN T cell responses to SARS-CoV-2 CD4 spike and nonspike peptide megapools. (D) Percentage of CD4+ and CD8+ TcR Vβ21.3 (black bars, left panels). An amount of 2.0%-3.9% of CD4+ T cells and 0.9%-2.6% of CD8+ T cells were TCR Vβ21.3+ among the 6 subjects studied. TCR Vβ21.3+ CD4+ T cells in 1 subject (16) showed a minor response to a SARS-CoV-2 CD4 nonspike peptide megapool. Each symbol represents an individual subject. Comparisons of the percentage of AIM+ T cells between unstimulated control and peptide megapool-stimulated cell cultures were tested by the Wilcoxon signed-rank test. Comparisons of the memory phenotypes of AIM+ T cells between subacute and follow-up visit were tested by the Mann-Whitney U test.

Discussion
This study addresses the T cell recognition of SARS-CoV-2 epitopes and the innate immune phenotype in MIS-C patients studied between March 2021 and March 2022. The magnitude of the T cell response in our MIS-C cohort possibly infected by SARS-CoV-2 Figure 6. T cell responses to peptide megapools derived from SARS-CoV-2 in MIS-C subjects healthy 6-14 months after the disease onset. Six MIS-C subjects (2, 3, 5, 12, 14, and 16) studied at their subacute phase were also studied 6-14 months later. Canonical CD4+ and CD8+, CD4− CD8− DN, and TCR Vβ21.3+ CD4+ and CD8+ T cell responses to SARS-CoV-2 peptide megapools were studied. The SI of AIM+ T cells from each individual subject was calculated to study the T cell responses. (A) CD4+ and CD8+ T cell responses to peptide megapools and their CCR6 expressions. Three subjects (2, 3, and 5) of the 6 subjects showed concurrent CD4+ and CD8+ T cell responses, and the other 3 follow-up MIS-C subjects (12, 14, and 16) showed only CD4+ T cell responses to SARS-CoV-2 (both CD4 spike (S) and nonspike (R) megapools). (B) Memory phenotypes of AIM+ CD4+ (left panels) and CD8+ (right panels) T cells. Red circles: subjects responding to both CD4 and CD8 epitopes (subjects 2, 3, and 5); blue circles: subjects responding to both CD4 spike and nonspike epitopes (subjects 12, 14, and 16). AIM+ CD4+ T cells showed a similar level of TEMRA, TEM, and TCM at follow-up visit compared with subacute MIS-C subjects (p > 0.05). AIM+ CD8+ T cells showed a slight increase in TEM (p > 0.05) and a similar level of TEMRA and TCM. (C) Percentage of DN T cells (left panel) and their responses to SARS-CoV-2 peptide megapools (right panels). Percentages of DN T cells were decreased in 5 subjects (2, 3, 5, 12, and 14) compared with subacute MIS-C subjects. One subject (3) showed DN T cell responses to SARS-CoV-2 CD4 and CD8 peptide megapools, and 2 subjects (14 and 16) showed DN T cell responses to SARS-CoV-2 CD4 spike and nonspike peptide megapools. (D) Percentage of CD4+ and CD8+ TcR Vβ21.3 (black bars, left panels). An amount of 2.0-3.9% of CD4+ T cells and 0.9-2.6% of CD8+ T cells were TCR Vβ21.3+ among the 6 subjects studied. TCR Vβ21.3+ CD4+ T cells in 1 subject (16) showed a minor response to a SARS-CoV-2 CD4 nonspike peptide megapool. Each symbol represents an individual subject. Comparisons of the percentage of AIM+ T cells between unstimulated control and peptide megapool-stimulated cell cultures were tested by the Wilcoxon signed-rank test. Comparisons of the memory phenotypes of AIM+ T cells between subacute and follow-up visit were tested by the Mann-Whitney U test.

Discussion
This study addresses the T cell recognition of SARS-CoV-2 epitopes and the innate immune phenotype in MIS-C patients studied between March 2021 and March 2022. The magnitude of the T cell response in our MIS-C cohort possibly infected by SARS-CoV-2 variants (Delta and Omicron) later in the pandemic was similar to the CD4+ Th response and the CD8+ CTL response that we reported in the first year of the pandemic [10]. Several subjects had only an antiviral CD4+ Th response, which in MIS-C seems to be unique, even though CD8+ CTLs were thought to be pathogenic by some investigators.
In fact, a correlation has been suggested between MIS-C disease severity and class I HLA A2, HLA B35, and HLA C4 that could present superantigens derived from the SARS-CoV-2 spike glycoproteins to specific oligoclonal TcRs carrying the Vβ21.3 chain, encoded by the TRBV11-2 gene [11]. Other authors found an oligoclonal expansion of Vβ21.3 T cells, which were also believed to be pathogenic [16]. We found numerous Vβ21.3 CD4+ and CD8+ T cells in our MIS-C cohort, but only in 1 subject CD8+ T cells recognized SARS-CoV-2 peptides, suggesting that the specificity is skewed to a different antigen or autoantigen. Evidence that healthy children studied 6 to 14 months after MIS-C still have numerous Vβ21.3 CD4+ and CD8+ T cells in circulation suggests that it is unlikely that these T cells are pathogenic but rather a characteristic of the T cell repertoire in these children.
Our data on the SARS-CoV-2-specific T cell response did not indicate a defect in the anti-viral-specific T cell repertoire, nor did we observe differences in the pattern of Th and CTL expansion in MIS-C subjects enrolled in 2020 [10], compared with MIS-C subjects enrolled in 2021 and 2022 and likely exposed to different variants of SARS-CoV-2. In this study, a high percentage of T cells, especially CD4+ T cells, expressed CCR6, which suggests trafficking to the endothelium, lungs, and gut that express CXCL20, the ligand for CCR6.
Consistent with our previous study, differences in the development of SARS-CoV-2specific T cell memory were observed with numerous effector and central memory T cells within the Th but not CTL compartment. Terminally differentiated effector T cells were abundant within the SARS-CoV-2-specific CD8+ but not CD4+ T cells, which may suggest a different timing of memory development.
A novel observation in this work is the antiviral function of DN T cells that recognize SARS-CoV-2 CD4+ and CD8+ peptide epitopes. DN T cells in the thymus become CD4+ CD8+ double positive (DP) to then mature as CD4+ or CD8+ single-positive, depending on the recognition of peptides presented by either MHC class II or class I molecules [17]. In adults, DN T cells are well described in systemic autoimmunity and via lymphokine secretion they contribute to the inflammatory milieu [18]. The DN T cells that we describe here are phenotypically different and recognize not only longer CD4 peptides but also short viral peptides contained in the CD8 peptide pools, suggesting recognition in association with both MHC class I and class II. Therefore, these DN T cells are already committed despite the absence of CD4 or CD8 coreceptors. Additionally, exhausted T cells may downregulate the CD4+ or CD8+ coreceptors under control of FAS signaling [19], but the expression of the IL-2 receptor CD25 that we reported associated with PD1 indicate functional T cell activation. In MIS-C, DN T cells in circulation could compensate for the severe lymphopenia in these patients; alternatively, DN T cells could have contributed to the inflammation by recognizing autoantigens. Evidence that DN T cells are virus specific and actually numerous in children that appeared to be nonresponders with canonical CD4+ and CD8+ T cells suggests that they are a mature T cell subset that participate in the antiviral response.
A sharp difference in MIS-C children enrolled in 2021-2022 versus children enrolled during the first year of the COVID-19 pandemic is the immune phenotype of the innate compartment: cDC2 and tmDC, tolerogenic, anti-inflammatory lineages, were low in our previous study [10] but numerous in this cohort.
We recognize both strengths and limitations to our study. We present a comprehensive characterization of the T cell response to SARS-CoV-2 and a detailed analysis of the innate APC compartment and enumeration and characterization of a specific TcR rearrangement that has been previously implicated in the inflammatory process. The novelty of finding numerous DN T cells, fully competent in responding to the virus and in the absence of DP T cells, undermines the paradigm of T cell selection, suggesting that in the absence of CD4+ and CD8+ coreceptors, the TcR is fully committed. A limitation is the small number of subjects studied and the lack of access to tissues to better define T cell homing and trafficking.
In summary, SARS-CoV-2-infected children who subsequently developed MIS-C showed a prevalent CD4+ Th response to the virus that reflected on the T cell memory phenotype where TEM and TCM cells were predominantly CD4, numerous DN T cells, and the activation of tolerogenic DC as CD14+ cDC2 and CD14+ CD11c+ CD11b+ ILT-4+ tmDC important to downsize the inflammation. Vb21.3+ T cells were detectable but did not recognize SARS-CoV-2 T cell epitopes. Over time, T cell epitope spreading occurred, as shown by the increased magnitude of the T cell responses and the expansion of T cell specificities in the late convalescent phase.

Study Populations
The study protocol for MIS-C and KD subjects was approved by the Institutional Review Board at the University of California San Diego (IRB #140220). Subjects were enrolled at Rady Children's Hospital, San Diego, following written parental informed consent and patient assent as appropriate. Twenty-two MIS-C subjects, 17 males and 5 females, aged 1.8 to 15 years were enrolled in the study from March 2021 to March 2022, 15-52 days after MIS-C onset to study SARS-CoV-2 T cell responses and to assess their innate immune phenotype. MIS-C patients' clinical and laboratory information at the time of hospital admission is described in Table 1. SARS-CoV-2 exposure was determined by PCR and antibody measurement. Blood samples were collected following intravenous immunoglobulin (IVIG) and other anti-inflammatory treatments 15-52 days after fever onset. Six of these subjects (2, 3, 5, 12, 14, and 16; Table 1) were studied at their follow-up visit 6-14 months after resolution of acute MIS-C symptoms.

Activation-Induced Markers (AIM) Assay and Enumeration of Vβ21.3 T Cells
Peripheral blood mononuclear cells (PBMCs) were separated from heparinized whole blood from MIS-C convalescent SARS-CoV-2 infected and KD subjects by Ficoll-Hypaque density centrifugation and frozen in liquid nitrogen. After thawing, 1 × 10 6 cells were stimulated in 96-well U-bottom plates with 1 µg/mL of different peptide megapools. PBMCs cultured with 0.1% DMSO, the same concentration of DMSO (solvent) in the megapool-stimulated cultures, served as unstimulated controls. After 24 h, cell cultures were harvested and stained with monoclonal antibodies and analyzed by flow cytometry Funding: This study has been funded by the NIH NIAID, RO1AI43586, to A.F.; by a grant from the Resources-Based Center for the Study of the Joint Microenvironment in Rheumatology to A.F.; by a grant from the Academic Senate of the University of California San Diego to A.F.; by the NIH NIAID, 75N9301900065, to A.S.; by the NHLBI, 3R01HL140898-03S1, to J.C.B. and A.H.T.; by the NICHD, 1R61HD105590, to J.C.B., and by the Gordon and Marilyn Macklin Foundation.
Institutional Review Board Statement: The Institutional Review Board at UCSD approved this study.

Informed Consent Statement:
The study protocol for MIS-C subjects was approved by the Institutional Review Board at the University of California San Diego (IRB #140220). The pediatric subjects were enrolled following written parental informed consent and patient assent as appropriate. The adult subjects were enrolled following written consent. Blood drawing of the pediatric subjects occurred following signature of the parental consent.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.